| Polyploidy is recognized as a common phenomenon in the evolution of plants. It is estimated that 50 to 80% of angiosperms are polyploids. The prominence of polyploidy in flowering plants implies that it has some adaptive significance. Polyploids often show novel phenotypes that are not present in their diploid progenitors or exceed the range of the contributing species. Some of these traits, such as increased drought tolerance, apomixis (asexual seed production), pest resistance, organ size and biomass, could allow polyploids to enter new niches or enhance their chances of being selected for use in agriculture. Many insights have emerged from recent explorations using laboratory generated or synthetic polyploids. Isatis indigotica Fort., the same family Cruciferae with Arabidopsis thaliana, is a prevalent Chinese medicinal herb. After selection for five years, tetraploid I. indigotica (2n=28) with better yield and enhanced resistance had been obtained, compared with diploid progenitor (2n=14). Previous study in chemical component analysis revealed that the content of anti-virus component in tetraploid I. indigotica was higher than that in diploid plant. Subsequent comparative expressive analysis using Arabidopsis thaliana whole genome Affymetrix gene chip (ATH1) indicated that there was variance in gene expression between tetraploid and diploid plant. In order to deepen the research in this area, and to lay a groundwork for plant breeding and increasing contents of fine chemical compounds by using bioengineering in the near future, cloning and characterization of seven genes involved in the signal transduction, the biosynthetic pathway of monolignol and transcriptional regulation are presented in this text for the first time. Among them, three genes encoding a novle leucine-rich repeat type receptor-like kinases (IiLTK) and two distinct calcium-dependent protein kinases (IiCDPK, IiCDPK2) participate in signal transduction;three genes encoding phenylalanine ammonia-lyase (IiPAL), caffeic acid O-methyltransferase (IiCOMT), caffeoyl CoA O-methyltransferase (IiCCoAOMT) participate in monolignol biosynthesis;one gene encoding gibberel (IiMYB) participates in transcriptional regulation.Leucine-rich repeat type receptor-like kinases (LRR-RLKs) play important roles in plant signal transduction. A novel protein kinase gene (designated as IiLTK) belonging toplant LRR-RLKs was cloned from tetraploid /. indigotica using RACE and PCR. Multiple sequence alignment showed that IiLTK protein had high homology to LRR-RLKs from other plant species. Besides, like other LRR-RLKs, the predicted IiLTK polypeptide included leucine rich repeats at the amino terminus, a transmembrane region and a serine/threonine protein kinase domain in the carboxyl terminus, which presented a typical structure of plant LRR-RLKs. Bioinformatic analysis of IiLTK genomic DNA revealed that it contained 16 exons and 15 introns. The 5' flanking region of IiLTK was isolated by genome walking technique, and some main as-acting elements including TATA box and stress-responsiveness elements were predicted and analyzed. The 3' flanking region of IiLTK was rich in A+T and it formed a stem structure, which was the typical feature of terminator. Semi-quantitative RT-PCR showed that the expression of IiLTK in root, stem and leaf of tetraploid plant was different compared with those in diploid progenitor. Further expression analysis reveals that the signaling components of defense/stress pathways, such as gibberellic acid (GA3), NaCl and Abscisic Acid (ABA), up-regulate the IiLTK transcription levels over the control. Cold treatment can also increase the expression of IiLTK, whereas methyl jasmonate (MeJA) treatment can decrease its expression.Plants have a novel kind of calcium-dependent protein kinase genes, which play important roles in plant development and especially in plant signal transduction. Two novel calcium-dependent protein kinase genes (designated as IiCDPK, UCDPK2) were cloned from tetraploid /. indigotica by using RACE and PCR. Analysis of IiCDPK and HCDPK2 genomic DNA revealed that IiCDPK contained 8 exons, 7 introns and the length of most exons was highly conserved;HCDPK2 contained 7 exons, 6 introns and the length of most exons was also highly conserved. The 5' flanking region of HCDPK2 was isolated by genome walking technique, and some main as-acting elements including TATA box and stress-responsiveness elements were predicted and analyzed. The 3' flanking region of HCDPK2 was rich in A+T and it formed a stem structure, which was the typical feature of terminator. The predicted IiCDPK and IiCDPK2 polypeptides included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs. IiCDPK and KCDPK2 were found to have extensivehomology with other plant CDPK proteins via multiple alignments. Semi-quantitative RT-PCR showed that the expression of IiCDPK and HCDPK2 in root, stem and leaf of tetraploid plant was different compared with those in diploid progenitor. Further expression analysis revealed that gibberellin (GA3), NaCl and cold treatments could up-regulate IiCDPK and HCDPK2 transcription. Methyl jasmonate (MeJA), Abscisic Acid (ABA) and Salicylic acid (SA) could only up-regulate IiCDPK transcription, while they had slight down-regulation effect on HCDPK2 transcription.Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the biosynthesis of monolignol. In the present study, we isolated a novel phenylalanine ammonia-lyase gene (designated as IiPAL) from tetraploid /. indigotica. Analysis of HPAL genomic DNA revealed that it was structurally similar to other plant PAL genes, with a single intron at a conserved position, and a long highly conserved second exon. The 5' flanking region of IiPAL was isolated by genome walking technique, and some main cw-acting elements including TATA box and stress-responsiveness elements were predicted and analyzed. Multiple sequence alignment showed that IiPAL protein had high homology to PALs from other plant species. Semi-quantitative RT-PCR revealed that the IiPAL expression in root and leaf from tetraploid sample was higher than that in diploid progenitor, whereas expression of IiPAL in stem was almost the same each other. Furthermore, the highest expression of IiPAL in tetraploid plant was found in root, which was found in stem in diploid plant. Further expression analysis revealed that gibberellin (GA3), abscisic acid (ABA), methyl jasmonate (MeJA) and cold treatments could up-regulate the IiPAL transcription in tetraploid plant.Caffeic acid O-methyltransferase is one of the key enzymes in monolignol biosynthesis. In the present study, we isolated a novel caffeic acid O-methyltransferase (designated as HCOMT) from tetraploid /. indigotica using RACE and PCR. Analysis of HCOMT genomic DNA revealed that it contained 4 exons and 3 introns. HCOMT was found to have extensive homology with other plant COMT proteins via multiple alignments. The amino acid sequence of HCOMT was highly homologous to the seven characteristic motifs of plant OMT-II protein. Furthermore, IiCOMT also shared high homology to two newly defined regions of the consensus sequence: motif SI, motif S2 respectively. Semi-quantitative RT-PCR revealed that the expression of IiCOMT in root,stem and leaf was all higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin (GA3), NaCl, methyl jasmonate (MeJA), Abscisic Acid (ABA), Salicylic acid (SA) and cold treatments could up-regulate IiCOMT transcription.Caffeoyl CoA O-methyitransferase is one of the key enzymes in monolignol biosynthesis. A novel caffeoyl CoA O-methyltransferase (designated as IiCCoAOMT) was cloned from tetraploid /. indigotica using RACE and PCR. The full-length cDNA of IiCCoAOMT was 1098 bp long with an open reading frame (ORF) of 774 bp encoding a polypeptide of 257 amino acid residues. Analysis of //CCo/40A/T genomic DNA revealed that IiCCoAOMT contained 5 exons and 4 introns. IiCCoAOMT was found to have extensive homology with other plant CCoAOMT proteins via multiple alignments and it had three characteristic motifs (Motif A, Motif B, Motif C) of plant OMT-I protein. Semi-quantitative RT-PCR revealed that the expression of IiCCoAOMT in root and leaf was higher in tetraploid sample than that in diploid progenitor, whereas expression of IiCCoAOMT in stem was almost the same each other. Further expression analysis revealed that gibberellin (GA3), NaCl, methyl jasmonate (MeJA), Abscisic Acid (ABA), Salicylic acid (SA) and cold treatments could up-regulate IiCCoAOMT transcription.A novel MYB-like transcription factor (designated as IiMYB) was cloned from tetraploid /. indigotica by using RACE and PCR. The full-length cDNA of IiMYB was 926 bp long with an open reading frame (ORF) of 600 bp encoding a polypeptide of 199 amino acid residues. Analysis of IiMYB genomic DNA revealed that IiMYB contained 3 exons and 2 introns. IiMYB was found to have high homology only with AtMYBL2 (Arabidopsis thaliana) and it also had only one MYB domain just like AtMYBL2. Semi-quantitative RT-PCR revealed that the expression of IiMYB in root, stem and leaf was all higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin (GA3), NaCl, methyl jasmonate (MeJA), Abscisic Acid (ABA) and Salicylic acid (SA) could up-regulate IiMYB transcription.As indicted in this text, the above-mentioned genes might have important functions and they have differential expression level in diploid and tetraploid /. indigotica, which might be the main reason for better yield and enhanced resistance in tetraploid plant compared with diploid plant. The above-mentioned genes and their characterization willprovide a solid base for further studies on prominence of polyploidy, at molecular and biochemical levels. The cloning of these genes also make it possible to provide some convenience for plant breeding and bioengineering fine chemicals in medicinal plant for human health in the future. |
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